A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

保护哺乳动物生殖系基因组完整性的新工艺

• 批准号: 10445720
• 负责人: Piroska Edit Szabó
• 金额: $ 51.9万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-20 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445720
• 关键词: Adopted; Affect; Affinity; B-DNA; BTB/POZ Domain; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biological Process; Cell Culture Techniques; Cells; Cellular biology; Chromosomal Rearrangement; Chromosome Deletion; Circular Dichroism; DNA; DNA Double Strand Break; DNA Methylation; DNA Modification Methylases; DNA Sequence Rearrangement; DNA Structure; DNA-Binding Proteins; Dependence; Development; EMSA; Enzymes; Epigenetic Process; Generations; Genetic; Genetic Models; Genome; Germ Cells; Germ Lines; Germ-Line Mutation; Grant; Hand; Human; Immunohistochemistry; In Vitro; Left; Location; Mammalian Cell; Maps; Measures; Methods; Methylation; Methyltransferase; Molecular; Monitor; Mus; Mutant Strains Mice; Nucleosomes; Organism; Population; Pregnancy; Process; Proteins; Purines; Pyrimidine; Refractory; Role; Short Tandem Repeat; Site; Spermatocytes; Tandem Repeat Sequences; Testing; Time; Transgenic Mice; Variant; Z-Form DNA; Zinc Fingers; base; epigenome; epigenomics; fetal; genome integrity; genome-wide; in vivo; male; mutant; mutation assay; next generation; novel; sperm cell

PROJECT SUMMARY Simple purine–pyrimidine (Pu/Py) repeats (PPRs) are known to form the left-handed, fragile Z-DNA structure. Such repeats are also known to be highly mutagenic, inducing large chromosomal deletions and rearrangements in the cells of higher organisms. The mutagenic effects of Z-DNA would be the most detrimental to a species if breaks occurred uncontrollably in the germ line and mutations occurring at unrepaired breaks were passed on to the next generation. We discovered a novel biological process in the mammalian germ line that controls Z- DNA structure at PPRs. Using the Zbtb43 mutant mouse genetic model and our transgenic mouse line that allows us to isolate germ cells, we found that a previously uncharacterized DNA binding protein, ZBTB43 remodels Z-DNA structure and protects from double-strand breaks in fetal male germ cells in vivo. By biochemical assays we found that ZBTB43 binds to PPR-rich DNA sites in the genome in vitro. ZBTB43 binding sites form Z-DNA and cause large genomic rearrangements in mammalian cells. By in vivo epigenome mapping we detected Z-DNA in mutant germ cells at the locations where ZBTB43 binding occurs in wild-type prospermatogonia. We hypothesize, therefore, that ZBTB43 safeguards genome integrity in the germ line by binding and eliminating Z-DNA at PPRs. In addition, we found that by eliminating Z-DNA, ZBTB43 promotes de novo methylation at PPRs during the time of global epigenetic remodeling. We propose to pursue the following Aims, using a combination of genetic, cell biology, biochemistry, and epigenomic approaches. In Aim 1, we will test the working hypothesis that ZBTB43 eliminates Z-DNA structure in vivo by directly binding to PPRs in fetal male germ cells. We will determine 1) the spatial and temporal changes of the Z-DNA structure in fetal male germ cells in the presence or absence of ZBTB43 protein in vivo; 2) the dependence of the Z-DNA remodeling process on the direct binding of ZBTB43 to the Z-DNA structure in vivo; and 3) the molecular requirements of ZBTB43 action on Z-DNA. In Aim 2, we will test the working hypothesis that ZBTB43 facilitates de novo DNA methylation in prospermatogonia indirectly by eliminating Z-DNA, thus revealing the sequences as substrates for de novo methyltransferases. We will map DNA methylation in the presence and absence of ZBTB43 during the epigenome remodeling process in fetal male germ cells. We will test whether de novo DNMTs methylate Z- DNA substrates in vitro. We will test whether ZBTB43 affects nucleosome occupancy at PPRs. In Aim 3, we will test the hypothesis that Z-DNA is mutagenic in the germ line and that ZBTB43 has evolved to manage that burden. We will map double-strand breaks in mutant fetal germ cells, test the anti-mutagenic effect of ZBTB43 in cell culture, and search for genomic rearrangements in sperm of Zbtb43 mutant males. By the end of the grant period, we will have identified and characterized the first example of how a DNA binding protein protects genome integrity and targets de novo DNA methylation by controlling Z-DNA structure in the mammalian germ line.

项目总结 简单的嘌呤-嘧啶(Pu/Py)重复序列(Pprs)形成了左手的、脆弱的Z-DNA结构。 众所周知，这种重复序列也是高度诱变的，会导致大量的染色体缺失和重排。 在高等有机体的细胞中。在以下情况下，Z-DNA的突变效应对物种的危害最大 在胚系中发生不可控制的断裂，并且在未修复的断裂处发生的突变被传递 献给下一代。我们在哺乳动物生殖系中发现了一种新的生物学过程，它控制着Z- Pprs的DNA结构。利用Zbtb43突变小鼠遗传模型和我们的转基因小鼠系 允许我们分离生殖细胞，我们发现了一种以前未鉴定的DNA结合蛋白，ZBTB43 在体内重塑Z-DNA结构并保护胎儿男性生殖细胞免受双链断裂。通过 生化检测发现，ZBTB43在体外能与基因组中富含PPR的DNA结合。ZBTB43结合 这些位点形成Z-DNA，并在哺乳动物细胞中引起大规模的基因组重排。通过活体表观基因组图谱 我们在野生型中ZBTB43结合的突变生殖细胞中检测到Z-DNA 繁茂的原细胞。因此，我们假设ZBTB43通过以下方式保护生殖系的基因组完整性 在PPR上结合和消除Z-DNA。此外，我们还发现，通过消除Z-DNA，ZBTB43促进了 在全球表观遗传重塑期间，Pprs的新甲基化。我们建议进行以下工作 目标，使用遗传学、细胞生物学、生物化学和表观基因组学方法的组合。在目标1中，我们将 验证ZBTB43通过直接与胎儿Pprs结合在体内消除Z-DNA结构的工作假说 雄性生殖细胞。我们将测定1)男性胎儿Z-DNA结构的空间和时间变化 存在或不存在ZBTB43蛋白的体内生殖细胞；2)Z-DNA重塑的依赖性 ZBTB43与体内Z-DNA结构直接结合的研究进展 ZBTB43对Z-DNA的作用。在目标2中，我们将检验ZBTB43促进从头DNA的工作假说 精原细胞中通过消除Z-DNA间接甲基化，从而揭示序列为底物 对于从头开始的甲基转移酶。我们将绘制在ZBTB43存在和不存在的情况下的DNA甲基化 胎儿男性生殖细胞的表观基因组重塑过程。我们将测试从头开始DNMTS甲基化Z- DNA底物的体外培养。我们将测试ZBTB43是否影响PPR的核小体占有率。在《目标3》中，我们将 测试Z-DNA在生殖系中是诱变的假设，以及ZBTB43已经进化到能够处理这一假设 负担。我们将绘制突变胎儿生殖细胞的双链断裂图谱，检测ZBTB43的抗突变作用 在细胞培养中，并寻找Zbtb43突变男性精子中的基因组重排。到赠款结束时 期间，我们将已经确定并表征了DNA结合蛋白如何保护基因组的第一个例子 通过控制哺乳动物生殖系中的Z-DNA结构的完整性和目标从头开始的DNA甲基化。